The present invention relates to novel compounds which make it possible to transfer nucleic acids into cells. More precisely, these novel compounds are lipid derivatives of polythiourea. They are useful for the in vitro, ex vivo or in vivo transfection of nucleic acids into various cell types.
With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It may involve the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.
Several methods have been proposed for the intracellular delivery of exogenous genetic material. One of them, in particular, is based on the use of nonviral vectors which constitute a highly advantageous alternative to the viral methods which are not completely risk free. These synthetic vectors have two main functions: to complex and to compact the nucleic acid to be transfected, and to promote its passage across the plasma membrane and possibly across the nuclear envelope.
Several families of synthetic vectors have thus been developed, such as for example polymers or alternatively biochemical vectors (consisting of a cationic protein combined with a cellular receptor ligand), but a major advance has in particular been made with the development of lipofectants and more particularly of cationic lipids. It has thus been demonstrated that cationic lipids, because of their overall positive charge, spontaneously interfere with DNA which is globally negative, forming nucleolipid complexes capable both of protecting the DNA against nucleases and of binding to the cellular membranes for intracellular release of the DNA.
Various types of cationic lipids have been synthesized to date: lipids comprising a quaternal ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE, and the like), lipopolyamines such as for example DOGS, DC-Chol or alternatively the lipopolyamines disclosed in Patent Application WO 97/18185, lipids combining both a quaternary ammonium group and a polyamine such as DOSPA, or alternatively lipids comprising various other cationic entities, in particular amidinium groups (for example ADPDE, ADODE or the lipids of patent application WO 97/31935).
However, the use of these cationic lipids as transfection agent still poses numerous problems, and their efficiency remains to be improved. In particular, it has been observed that to obtain efficient and stable nucleolipid complexes, it is in general necessary for these complexes to be highly cationic. However, it would be desirable to be able to have available vectors which are not cationic so as to form, with the nucleic acid, particles which are globally neutral or negative. Indeed, it has been observed that the globally cationic complexes formed between the nucleic acid and the cationic lipids tend to be captured by the reticuloendothelial system, which induces their elimination. In addition, the plasma proteins tend to become adsorbed at their surface because of the overall positive charge of the complexes formed, and this results in a loss of the transfection power. Furthermore, in a context of local injection, the presence of a large overall positive charge prevents the diffusion of the nucleic acid complexes away from the site of administration because the complexes become adsorbed onto the extracellular matrices; the complexes can therefore no longer reach the target cells, which consequently causes, a decrease in the transfer efficiency in relation to the injected quantity of complexes. Finally, it has also been observed, in many instances, that cationic lipids have an inflammatory effect.
The object of the present invention is precisely to provide novel transfecting compounds which are innovative by virtue of their polythiourea functional group and which are capable of being efficiently used for the in vitro, ex vivo or in vivo transfection of nucleic acids. These novel compounds are particularly advantageous because:
the absence of positive charges from their structure makes it possible to solve the many problems raised by the use of cationic vectors discussed above,
just like cationic lipids, they are capable of complexing and compacting nucleic acids and of promoting their transfection.
A first subject of the present invention is thus transfecting compounds characterized in that they consist of a polythiourea part linked to a lipid via a spacer.
In particular, the subject of the present invention is transfecting compounds of general formula (I): 
in which:
l is an integer chosen from 0 and 1,
n is an integer chosen from 1, 2, 3, 4, 5 and 6,
m is an integer chosen from 2, 3 and 4, it being possible for m to take different values within the different groups xe2x80x94[NHxe2x80x94CSxe2x80x94NHxe2x80x94(CH)m]xe2x80x94,
Rxe2x80x2 represents a group of general formula (II): 
xe2x80x83in which q is an integer chosen from 1, 2, 3, 4, 5 and 6, and p is an integer chosen from 2, 3 and 4, it being possible for p to take different values within the different groups xe2x80x94[(CH2)pxe2x80x94NHxe2x80x94CSxe2x80x94NH]xe2x80x94,
R represents either a hydrogen atom or a group of general formula (II) as defined above, it being understood that when n is 1 and l is 0, then at least one group R is of formula (II),
X, in the formulae (I) and (II), represents a saturated or unsaturated, linear or cyclic aliphatic group, comprising 1 to 8 carbon atoms, a mercaptomethyl (xe2x80x94CH2SH) group, or alternatively a hydrophilic chain chosen from the groups:
xe2x80x94(CH2)xxe2x80x94(CHOH)uxe2x80x94H with x an integer chosen from 1 to 10 and u an integer chosen from 1, 2, 3, 4, 5 and 6, or alternatively,
xe2x80x94(OCH2CH2O)vxe2x80x94H with v an integer chosen from 1, 2 and 3, it being understood that no more than one substituent X, both in the formulae (I) and (II), represents a hydrophilic chain,
Y represents a spacer,
and L represents:
either a group xe2x80x94N(R1)R2 with R1 and R2 which represent, independently of each other, a hydrogen atom or alternatively a fatty aliphatic chain, or alternatively a group of formula xe2x80x94(CH2)txe2x80x94OZ with t representing an integer chosen from 11, 12, 13, 14 or 15 and Z represents a sugar, a polyol or a PEG, it being understood that at least one of R1 and R2 is different from hydrogen,
or a group xe2x80x94OR3, with R3 which represents a steroid derivative.
According to the present invention, the term xe2x80x9cspacerxe2x80x9d is understood to mean any chemical group which makes it possible both to provide the linkage between the polythiourea part and the lipid part of the molecule, and to keep these two parts apart so as to attenuate any undesirable interruption between them. Preferred spacers may for example consist of one or more chemical functional groups chosen from alkyls having 1 to 6 carbon atoms, ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or else aromatic rings. For example, the spacer may be chosen from the groups of formula:
xe2x80x94NHxe2x80x94C(O)xe2x80x94CH2xe2x80x94CH2xe2x80x94
or:
xe2x80x94(CH2xe2x80x94)ixe2x80x94Wxe2x80x94(CH2)jxe2x80x94
in which i and j are integers chosen between 1 and 6 inclusive and W is a group chosen from ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or alternatively aromatic rings.
For the purposes of the present invention, the expression xe2x80x9cfatty aliphatic chainsxe2x80x9d is understood to mean alkyl groups containing 10 to 22 carbon atoms which are saturated or unsaturated and optionally containing one or more heteroatoms, provided that said fatty aliphatic chains exhibit lipid properties. Preferably, they are linear or branched alkyl groups containing 10 to 22 carbon atoms and 1, 2 or 3 unsaturations. Preferably, said alkyl groups comprise 10, 12, 14, 16, 18, 20 or 22 carbon atoms. There may be mentioned more particularly the aliphatic groups xe2x80x94(CH2)11CH3, xe2x80x94(CH2)13CH3, (CH2)15CH3 and xe2x80x94(CH2)17CH3.
The term xe2x80x9csugarxe2x80x9d is understood to mean, for the purposes of the invention, any molecule consisting of one or more saccharides. There may be mentioned, by way of example, sugars such as pyranoses and furanoses, for example glucose, mannose, rhamnose, galactose, fructose or alternatively maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorose, melibiose, and the like. Preferably, the sugar(s) are chosen from glucose, mannose, rhamnose, galactose, fructose, lactose, saccharose and cellobiose. Furthermore, it may also involve so-called xe2x80x9ccomplexxe2x80x9d sugars, that is to say several sugars which are covalently coupled to each other, each sugar being preferably chosen from the list cited above. As suitable polysaccharides, there may be mentioned dextran, xcex1-amylose, amylopectin, fructans, mannans, xylans and arabinans. Some preferred sugars may in addition interact with the cell receptors, such as for example certain types of lectin.
According to the invention, the term xe2x80x9cpolyolxe2x80x9d is also understood to mean any linear, branched or cyclic hydrocarbon molecule comprising at least two hydroxyl functional groups. There may be mentioned by way of example glycerol, ethylene glycol, propylene glycol, tetritols, pentitols, cyclic pentitols (or quercitols), hexitols such as mannitol, sorbitol, dulcitols, cyclic hexitols or inositols, and the like (Stanek et al., The Monosaccharides Academic Press, pp. 621-655 and pp. 778-855). According to a preferred aspect; the polyols are chosen from the alcohols of general formula: 
for which s is chosen from 2, 3, 4, 5 and 6.
When the compounds of general formula (I) according to the invention contain a polyethylene glycol (PEG) group, the latter generally comprises between 2 and 120 xe2x80x94OCH2CH2Oxe2x80x94 units, and preferably between 2 and 80 xe2x80x94OCH2CH2Oxe2x80x94 units. This may include simple PEGs, that is to say whose chain ending ends with a hydroxyl group, or else PEG whose terminal group is chosen from alkyls, for example methyl.
For the purposes of the present invention, the expression xe2x80x9csteroid derivativesxe2x80x9d is understood to mean polycyclic compounds of the cholestane type. These compounds may be natural or otherwise and are more preferably chosen from cholesterol, cholestanol, 3-xcex1-5-cyclo-5-xcex1-cholestan-6-xcex2-ol, cholic acid, cholesteryl formate, chotestanyl formate, 3xcex1,5-cyclo-5xcex1-cholestan-6xcex2-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5a-dimethylhexadecahydrocyclopenta[a]cyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-ylamine, or cholestanylamine.
According to a preferred variant of the invention, the transfecting compounds have the general formula (lII): 
in which X, m, n and Y are as defined above in general formula (I), with the exception of n which is different from 1, and R1 and R2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of R1 and R2 is different from hydrogen.
More preferably still, the transfecting compounds of the invention have the general formula (IV): 
in which m, n and Y are as defined above in general formula (I), with the exception of n which is different from 1, and R1 and R2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of R1 and R2 is different from hydrogen.
It is understood that the present invention also relates to the isomers of the products of general formula (I) when they exist, as well as mixtures thereof.
The preparation of the compounds of general formula (I) according to the present invention is carried out using the following steps, in the order presented or according to any other known and equally suitable variant, using conventional organic synthesis techniques, in solution or on solid supports, which are well known to a person skilled in the art:
1) Production of the Lipid Part L
When the lipid part L of the compounds of general formula (I) is represented by a group xe2x80x94N(R1)R2 with R1 and/or R2 which represent a fatty aliphatic chain, the amine of formula HN(R1)R2 is first of all formed. Said amine may be obtained by condensing a carboxylic acid and an amine, one containing the substituent R1 and the other the substituent R2, to form the corresponding amide, followed by reduction of said amide thus obtained.
Amide formation is advantageously carried out by mixing constituents and melting, by heating at a temperature of greater than the melting point of the substances involved, in general between 20xc2x0 C. and 200xc2x0 C., followed by elimination of the water produced by dehydrating the medium; or more advantageously in the presence of a desiccating agent such as for example phosphorus pentoxide or any other substance which can absorb water. The formation of this intermediate amide may also be carried out using a variant of this method or another method for forming an amide (such as for example peptide-coupling type) involving carboxylic acids or derivatives thereof, and varying conditions and reagents [R. C. Larock, Comprehensive Organic Transformations, VCH Publishers] well known to a person skilled in the art.
The reduction of the amide previously obtained to an amine of formula HN(R1)R2 may be carried out for example using a reducing agent such as lithium aluminum hydride, or any other hydride or reducing agent effective in this case. The procedure is then preferably carried out in an aprotic solvent (for example tetrahydrofuran or ethers) at a temperature below the boiling point of the solvent or under a dry and/or inert atmosphere.
According to another variant, the lipid part designated as HN(R1)R2 may be commercially available.
When R1 and/or R2 represent(s) a group of formula xe2x80x94(CH2)txe2x80x94OZ, the procedure is carried out as described above for forming the alkyl part, followed by simple coupling with a commercial PEG, polyol or sugar according to conventional techniques known to a person skilled in the art.
When the lipid part L of the compounds of the general formula (I) is represented by a group xe2x80x94OR3, the latter is preferably chosen from commercially available products.
2) Grafting of the Spacer Y
The spacer Y is then attached to the lipid part L obtained in the preceding stage according to conventional techniques known to a person skilled in the art. According to a preferred variant, an amide bond is made by N-acylation of the lipid part L in an appropriate solvent such as dichloromethane, chloroform, tetrahydrofuran, or any other ether, at a temperature below the boiling point of the solvent, and under a dry and/or inert atmosphere. This reaction is preferably carried out in the presence of an amine-containing base such as N,N-dimethylaminopyridine, or in the presence of this base mixed with non-nucleophilic amine-containing bases such as triethylamine or else ethyl diisopropylamine. Pyridine may also be used, alone or mixed with another base, diluted with one of the solvents mentioned or used itself as solvent.
3) Formation of the Polythiourea Chain
The third part of the synthesis of the compounds of general formula (I) consists in the successive introduction of the thiourea units. This will be carried out in a series of reactions which may be repeated as many times as necessary in order to obtain the desired polythiourea part. According to a preferred method, the procedure is carried out in the following manner:
A) There is first of all grafted onto the Yxe2x80x94C(O)xe2x80x94L obtained in the preceding stage the first part of the unit in the form of a member xe2x80x94HNxe2x80x94(CHR)mxe2x80x94 group. For that, the procedure is advantageously carried out starting with a diamine-containing member of formula H2Nxe2x80x94(CHR)mxe2x80x94NH2 in the presence of a coupling agent, for example 1-benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 1-benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-(1H-benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate or tetrafluoroborate (HBTU or TBTU), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or else 1-(3-trimethylammoniopropyl)-3-ethylcarbodiimide iodide, supported or otherwise. This coupling is carried out in a suitable solvent, for example dichloromethane, chloroform, tetrahydrofuran or any other ether, at a temperature below the boiling point of a solvent, and under a dry and/or inert atmosphere. The procedure is also carried out in the presence of a non-nucleophilic amine-containing base, for example ethyldiisopropylamine, triethylamine or else triisopropylamine. If the nature of the lipid part and of the spacer is compatible, a sequence of the SCNxe2x80x94(CHR)mxe2x80x94 type, or a precursor, may be grafted, thus making it possible to continue the synthesis through a stage such as that described below in C),
B) The product obtained in the preceding stage is then converted, according to a preferred technique, to isothiocyanate by treating with carbon disulfide (CS2), or with any other reagent known to the person skilled in the art for obtaining such a functionality [H. Ulrich, Chemistry and Technology of Isocyanates, Wiley (1996). The Chemistry of Cyanates and their Thio Derivatives, S. Patai Ed., Wiley (1977). S. Ozaki, Recent Advances in Isocyanate Chemistry, Chem. Rev. 72, 457 (1972)]. The reaction is advantageously carried out in a solvent such as for example tetrahydrofuran, or any other compatible ether solvent, at a temperature varying between that of the cooling mixtures and about 20xc2x0 C. The procedure is also carried out in the presence of an agent capable of promoting the reaction and/or of trapping the hydrogen sulfide released during the reaction, for example dicyclohexylcarbodiimide (DCC).
C) The thiourea unit is then formed from the isothiocyanate obtained in the preceding stage so as to allow, where appropriate, the introduction of another segment of formula xe2x80x94CHR)mxe2x80x94. Advantageously, a diamine of formula H2Nxe2x80x94(CHR)mxe2x80x94NH2, optionally protected, is reacted, in its neutral form or in the form of an acid salt, with the isothiocyanate obtained in the preceding stage. This reaction is optionally carried out in the presence of a non-nucleophilic amine-containing base, for example triethylamine, ethyldiisopropylamine, triisopropylamine or else 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The procedure is preferably carried out in a suitable solvent such as dichloromethane, chloroform, tetrahydrofuran or any other compatible ether or solvent, at a temperature which may be between that of the cooling mixtures and the reflux temperature of the solvent.
Stages B) and C) described above are then repeated sequentially and in the required order until the desired structure is obtained, so as to introduce the desired unit in n copies. To obtain branched structures, the procedure is carried out in a similar manner by introducing, at the appropriate time, the molecule(s) required to obtain a substitution R as described by formula (II).
4) Ending of the Polythiourea Part by Introducing the Substituent X
The last stage allowing the ending of the polythiourea-type chain(s) consists in introducing the substituent X. For that, conventional grafting methods known to a person skilled in the art, chosen according to the nature of the substituent X, are used. For example, when X represents an alkyl, the procedure is carried out by reacting an alkyl isothiocyanate, in the presence, when necessary, of a non-nucleophilic amine-containing base such as for example triethylamine, ethyldiisopropylamine, triisopropylamine or else 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction is performed in a suitable solvent, for example dichloromethane, chloroform, tetrahydrofuran or any other compatible ether, at a temperature between the temperature of the cooling mixtures and the reflux temperature of the solvent.
Naturally, when the various substituents can interfere with the reaction, it is preferable to protect them beforehand with compatible radicals which can be put in place and removed without affecting the remainder of the molecule. For that, the procedure is carried out according to conventional methods known to a person skilled in the art, and in particular according to the methods described in T. W. Greene, Protective Groups in Organic Synthesis, Wiley-lnterscience, in McOmie, Protective Groups in Organic Chemistry, Plenum Press, or in P. J. Kocienski, Protecting Groups, Thieme.
Moreover, each stage of the method of preparation may be followed, where appropriate, by stages for separating and purifying the compound obtained according to any method known to a person skilled in the art.
Preferred compounds according to the present invention are:
The 3-(2-{3-[2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]thioureido}ethyl)-1-methylthiourea, designated herein as DTTU or as DT-3TU, corresponds to the general formula (I), wherein X=xe2x80x94CH3; m=2; R=H; n=3; l=0; Y=NHxe2x80x94COxe2x80x94CH2xe2x80x94CH2; and L=xe2x80x94N(R1)R2 où R1=R2=C14H29. Designation of this compound as DT-3TU, refers to the three thiourea groups comprised therein; in addition, examples of this nomenclature include, DT-4TU comprising four thioureas, DT-2TU comprising two thioureas, etc.
The 3-(2-{3-[2-(3-{2-[3-(2-{3-[ditetradecyl-carbamoyl]propionylamino}-ethyl)-thioureido]-ethyl}-thioureido)-ethyl]-thioureido}-ethyl)-1-methylthiourea or DT-4TU is according to the general formula (I), wherein X=xe2x80x94CH3; m=2; R=H; n=4; and l==0; Y=NHxe2x80x94COxe2x80x94CH2xe2x80x94CH2; and L=xe2x80x94N(R1)R2 where R1=R2=C14H29.
The DT-3TU diol or Synthesis of [2-(3-{2-[3-(2-{3-[2-(3-(ditetradecyl-carbamoyl)propionylamino)-ethyl]-thioureido}ethyl)-thioureido]-ethyl}-thioureido)-ethyl]-propane-1,2-diol, is according to the general formula (I), wherein: 
m=2
R=H
n=3
l=0
Y=NHxe2x80x94COxe2x80x94CH2xe2x80x94CH2; and
L=xe2x80x94N(R1)R2 where R1=R2=C14H29.
The DT-2TU diol or [2-(3-{2-[3-(ditetradecyl-carbamoyl)propionylamino]-ethyl}-thioureido)-ethyl]-propane-1,2-diol Where according to the general formula (I), wherein 
m=2
R=H
n=2
l=0
Y=NHxe2x80x94COxe2x80x94CH2xe2x80x94CH2; and
L=xe2x80x94N(R1)R2 where R1=R2=C14H29 
Another subject of the invention relates to the compositions comprising a transfecting compound according to the invention and a nucleic acid. The respective quantities of each component may be easily adjusted by a person skilled in the art according to the transfecting compound used, the nucleic acid and the desired applications (in particular the type of cells to be transfected).
For the purposes of the invention, the expression xe2x80x9cnucleic acidxe2x80x9d is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They may be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences such as DNA/RNA chimeroplasts or synthetic or semisynthetic sequences, and oligonucleotides which are modified or otherwise. These nucleic acids may be of human, animal, plant, bacterial or viral origin and the like. They may be obtained by any technique known to persons skilled in the art, and in particular by the screening of libraries, by chemical synthesis or by mixed methods including the chemical or enzymatic modification of sequences obtained by the screening of libraries. They may be chemically modified. In general, they contain at least 10, 20, 50 or 100 consecutive nucleotides, and preferably at least 200 consecutive nucleotides. More preferably still, they contain at least 500 consecutive nucleotides.
As regards more particularly deoxyribonucleic acids, they may be single- or double-stranded, as well as short oligonucleotides or longer sequences. In particular, the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes and the like. These deoxyribonucleic acids may carry a prokaryotic or eukaryotic replication origin which is functional or otherwise in the target cell, one or more marker genes, sequences for regulating transcription or replication, genes of therapeutic interest, anti-sense sequences which are modified or otherwise, regions for binding to other cellular components, and the like.
Preferably, the nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences, for example one or more promoters and a transcriptional terminator which are active in the target cells.
For the purposes of the invention, the expression gene of therapeutic interest is understood to mean in particular any gene encoding a protein product having a therapeutic effect. The protein product thus encoded may in particular be a protein or a peptide. This protein product may be exogenous, homologous or endogenous in relation to the target cell, that is to say a product which is normally expressed in the target cell when the latter has no pathological condition. In this case, the expression of a protein makes it possible, for example, to palliate an insufficient expression in the cell or the expression of a protein which is inactive or weakly active because of a modification, or to overexpress said protein. The gene of therapeutic interest may also encode a mutant of a cellular protein, having increased stability, modified activity and the like. The protein product may also be heterologous in relation to the target cell. In this case, an expressed protein may, for example, supplement or provide an activity which is deficient in the cell, allowing it to combat a pathological condition, or to stimulate an immune response.
Among the therapeutic products for the purposes of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines and cytokines as well as their inhibitors or their antagonists: interleukins, interferons, TNF, antagonists of interleukin 1, soluble receptors for interleukin 1 or TNFxcex1, and the like (FR 92/03120), growth factors, neuro-transmitters or their precursors or synthesis enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP/pleiotrophin and the like), apolipoproteins (ApoAI, ApoAIV, ApoE, and the like, FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, Rap1A, DCC, k-rev, and the like, FR 93/04745), genes encoding factors involved in coagulation (Factors VII, VII, IX), the genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), the genes for hemoglobin or other protein carriers, metabolic enzymes, catabolic enzymes and the like.
The nucleic acid of therapeutic interest may also be a gene or an anti-sense sequence or a DNA encoding an RNA with ribosome function, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed in the target cell into RNAs which are complementary to cellular mRNAs and thus block their translation to protein, according to the technique described in Patent EP 140 308. The therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).
As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide, which is capable of generating an immune response in humans or in animals. In this specific embodiment, the invention allows the production of vaccines or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular for treating or preventing infections, for example viral or bacterial infections, or cancerous states. They may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the syncitia forming virus, other viruses, or antigenic peptides specific for tumors (EP 259 212).
Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and/or the gene encoding the antigenic peptide in the desired cell or organ. They may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences derived from the genome of a virus. In this regard, there may be mentioned, for example, the promoters of the E1A, MLP, CMV and RSV genes, and the like. In addition, these expression sequences may be modified by the addition of activating or regulatory sequences, and the like. The promoter may also be inducible or repressible.
Moreover, the nucleic acid may also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also comprise a signal sequence directing the synthesized therapeutic product towards a particular compartment of the cell.
The compositions according to the invention may, in addition, comprise one or more adjuvants capable of combining with the transfecting compound/nucleic acid complexes and of improving the transfecting power thereof. In another embodiment, the present invention therefore relates to compositions comprising a nucleic acid, a transfecting compound as defined above and at least one adjuvant capable of combining with the transfecting compound/nucleic acid complexes and of improving the transfecting power thereof. The presence of this type of adjuvant (lipids, peptides, proteins or polymers for example) may make it possible advantageously to increase the transfecting power of the compounds. In this regard, the compositions of the invention may comprise, as adjuvant, one or more neutral lipids, which possess in particular the property of forming lipid aggregates. The term xe2x80x9clipid aggregatexe2x80x9d is a generic term which includes liposomes of any type (both unilamellar and multilamellar) as well as micelles or else more amorphous aggregates.
More preferably, the neutral lipids used within the framework of the present invention are lipids containing two fatty chains. In a particularly advantageous manner, natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions are used. They may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oteoylpalmitoyl-phosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidyl-ethanolamines as well as their derivatives which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or asialogangliosides (such as in particular asialoGM1 and GM2). Advantageously, the lipid adjuvants used in the context of the present invention are chosen from DOPE, DOPC or cholesterol.
These different lipids may be obtained either by synthesis or by extraction from organs (for example the brain) or from eggs, by conventional techniques well known to persons skilled in the art. In particular, the extraction of the natural lipids may be carried out by means of organic solvents (see also Lehninger, Biochemistry).
Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvants for one equivalent of nucleic acid in mol/mol and, more preferably, from 0.5 to 5 molar equivalents.
According to another alternative, the adjuvants mentioned above making it possible to improve the transfecting power of the compositions according to the present invention, in particular the peptides, proteins or certain polymers, such as polyethylene glycol, may be conjugated with the transfecting compounds according to the invention, and not simply mixed. In this case, they are covalently linked either to the substituent X in the general formula (I), or to the end of the alkyl chain(s) R1 and/or R2 when the latter are fatty aliphatic chains. It is also advantageous to use as adjuvant, a polyethylene glycol covalently linked to cholesterol (chol-PEG). In effect, when such adjuvant is used with transfectant compositiosn according to the present invention, resulting particles have a smaller size, thereby decreasing aggregation thereof, and increasing their half-life in the blood circulation. Amount of transfectant DT-3TU used according to the present invention is such that particles have a size inferior to 500 nm. Preferred amount of DT-3TU used is at least equal to 40 nmol of lipids DT-3TU/xcexcg of DNA (See Examples 11, 13, and 14 herein below).
According to a particularly advantageous embodiment, the compositions of the present invention comprise, in addition, a targeting element which makes it possible to orient the transfer of the nucleic acid. This targeting element may be an extracellular targeting element which makes it possible to orient the transfer of the nucleic acid toward certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells and the like). It may also be an intracellular targeting element which makes it possible to orient the transfer of the nucleic acid toward certain preferred cellular compartments (mitochondria, nucleus and the like). The targeting element may be mixed with the transfecting compounds according to the invention and with the nucleic acids, and in this case, the targeting element is preferably covalently linked to a fatty alkyl chain (at least 10 carbon atoms) or to a polyethylene glycol. According to another alternative, the targeting element is covalently linked to the transfecting compound according to the invention either at the level of the substituent X or on the spacer Y, or else at the end of R1 and/or R2 when the latter represent fatty aliphatic chains. Finally, the targeting element may also be linked to the nucleic acid as was specified above.
Among the targeting elements which may be used within the framework of the invention, there may be mentioned sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof. Preferably, they are sugars, peptides, vitamins or proteins such as for example antibodies or antibody fragments, ligands of cell receptors or fragments thereof, receptors or receptor fragments. For example, they may be ligands of growth factor receptors, cytokine receptors, cellular lectin-type receptors, folate receptors, or RGD sequence-containing ligands with an affinity for the receptors for adhesion proteins such as the integrins. There may also be mentioned the receptors for transferin, HDLs and LDLs, or the folate transporter. The targeting element may also be a sugar which makes it possible to target lectins such as the receptors for asialoglycoproteins or for sialydes, such as the Sialyl Lewis X, or alternatively an Fab fragment of antibodies, or a single-chain antibody (ScFv).
The subject of the invention is also the use of the transfecting compounds as defined above for transferring nucleic acids into cells in vitro, in vivo or ex vivo. More precisely, the subject of the present invention is the use of the transfecting compounds according to the invention for the preparation of a medicament intended for treating diseases, in particular diseases resulting from a deficiency in a protein or nucleic product. The polynucleotide contained in said medicament encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo.
For uses in vivo, for example in therapy or for studying the regulation of genes or the creation of animal models of pathological conditions, the compositions according to the invention can be formulated for administration by the topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intra-muscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal route, and the like. Preferably, the compositions of the invention contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular a direct injection into the desired organ, or for administration by the topical route (on the skin and/or the mucous membrane). They may be in particular isotonic sterile solutions, or dry, in particular freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, allow the constitution of injectable solutions. The nucleic acid doses used for the injection as well as the number of administrations may be adapted according to various parameters, and in particular according to the mode of administration used, the relevant pathological condition, the gene to be expressed, or the desired duration of treatment. As regards more particularly the mode of administration, it may be either a direct injection into the tissues, for example at the level of the tumors, or an injection into the circulatory system, or a treatment of cells in culture followed by their reimplantation in vivo by injection or transplantation. The relevant tissues within the framework of the present invention are, for example, the muscles, skin, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bones, cartilages, pancreas, kidneys, bladder, stomach, intestines, testicles, ovaries, rectum, nervous system, eyes, glands, connective tissues, and the like.
Another subject of the present invention relates to a method of transferring nucleic acids into cells comprising the following steps:
(1) bringing the nucleic acid into contact with a transfecting compound according to the present invention, to form a complex, and
(2) bringing the cells into contact with the complex formed in (1).
The invention relates, in addition, to a method of treating the human or animal body comprising the following steps:
(1) bringing the nucleic acid into contact with a transfecting compound according to the present invention, to form a complex, and
(2) bringing the cells of the human or animal body into contact with the complex formed in (1).
The cells may be brought into contact with the complex by incubating the cells with said complex (for uses in vitro or ex vivo), or by injecting the complex into an organism (for uses in vivo). In general, the quantity of nucleic acid intended to be administered depends on numerous factors such as for example the disease to be treated or to be prevented, the actual nature of the nucleic acid, the strength of the promoter, the biological activity of the product expressed by the nucleic acid, the physical condition of the individual or of the animal (weight, age and the like), the mode of administration and the type of formulation. In general, the incubation is preferably carried out in the presence, for example, of 0.01 to 1000 xcexcg of nucleic acid per 106 cells. For administration in vivo, nucleic acid doses ranging from 0.01 to 50 mg may for example be used. The administration may be carried out as a single dose or repeated at intervals.
In the case where the compositions of the invention contain, in addition, one or more adjuvants as defined above, the adjuvant(s) may be mixed beforehand with the transfecting compound according to the invention and/or the nucleic acid. Alternatively, the adjuvant(s) may be administered before the administration of the nucleolipid complexes.
According to another advantageous alternative, the tissues may be subjected to a chemical or physical treatment intended to improve the transfection. In the case of the physical treatment, the latter may use electrical pulses as in the case of electrotransfer, or else mechanical forces as in the case of sodoporation.
The present invention thus provides a particularly advantageous method for transferring nucleic acids in vivo, in particular for the treatment of diseases, comprising the in vivo or in vitro administration of a nucleic acid encoding a protein or which can be transcribed into a nucleic acid capable of correcting said disease, said nucleic acid being combined with a transfecting compound according to the invention under the conditions defined above.
The transfecting compounds of the invention are particularly useful for transferring nucleic acids into primary cells or into established lines. They may be fibroblast cells, muscle cells, nerve cells (neurons, astrocytes, glial cells), hepatic cells, hematopoietic cells (lymphocytes, CD34, dendritic cells, and the like), epithelial cells and the like, in differentiated or pluripotent form (precursors).
Another subject of the present invention also relates to the transfection kits which comprise one or more transfecting compounds according to the invention and/or mixtures thereof. Such kits may be provided in the form of a packaging which is compartmented so as to receive various containers such as for example vials or tubes. Each of these containers comprises the various elements necessary to carry out the transfection, individually or mixed: for example one or more transfecting compounds according to the invention, one or more nucleic acids, one or more adjuvants, cells, and the like.
In addition to the preceding arrangements, the present invention also comprises other characteristics and advantages which will emerge from the examples and figures below, which should be considered as illustrating the invention without limiting its scope. In particular, the applicant proposes, without limitation, an operating protocol as well as reaction intermediates which may be used to prepare the transfecting compounds according to the invention. Of course, it is within the capability of persons skilled in the art to draw inspiration from this protocol or intermediate products to develop similar methods so as to arrive at these same compounds.
EtBr: ethidium bromide
DCC: dicyclohexylcarbodiimide
DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DTTU: 3-(2-{3-[2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]-ethyl}thioureido)ethyl]thioureido}ethyl)-1-methylthiourea (also designated DT-3TU)
EPC: L-xcex1-phosphatidylcholine 95% (egg)
PyBOP: benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
TBE: tris-borate-EDTA
TFA: trifluoroacetic acid
THF: tetrahydrofuran